Stabilization of enzymes

ABSTRACT

Enzyme-containing formulations having improved stability and enzymatic activity in liquid medium, comprising one or more protease enzymes produced from any Bacillus bacteria, at least about 5% alkali metal halide salt, and at least about 50% polyol. Also disclosed are methods for making such formulations.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to enzyme compositions and liquidformulations including enzymes. Particularly, the invention relates tomethods for stabilizing enzymes and to liquid formulations with thestabilized enzymes.

[0003] 2. Background

[0004] The stabilization of enzymatic activity is a standing problem inall areas of technology where enzymes are likely to be applied.Stability in this sense includes resistance to decrease in enzymaticactivity prior to usage, e.g., under storage conditions. Stabilizationof enzymes in liquid formulations is particularly a problem For example,a pre-formulated liquid enzyme concentrate may sometimes be stored forweeks or months before eventually being blended into a final product(e.g., a personal care product, such as a hand cream; or a cleaningproduct, such as a liquid detergent). Similarly, formulated liquidproducts containing enzymes may sit in storage for lengthy periods oftime before use, as well. For a variety of reasons, the activity ofenzymes in liquid formulations typically decreases over time.

[0005] The prior art has attempted to deal with this problem. Forexample, organic compounds, such as sodium formate, and propylene glycolor glycerol are often added to liquid enzyme formulations. U.S. Pat. No.4,318,818, Letton et al., issued Mar. 9, 1982, discloses liquiddetergents containing enzymes and an enzyme-stabilizing systemcomprising calcium ions and a low molecular weight carboxylic acid orsalt, preferably a formate. The composition preferably contains ananionic surfactant and a saturated fatty acid. U.S. Pat. No. 4,404,115,Tai, issued Sep. 13, 1983, discloses cleaning compositions containingenzymes, alkali metal pentaborate, al metal sulfite and a polyol. Whilesomewhat effective, additives such as formate and other organic saltsunfortunately are costly and, thus, significantly add to the expense ofthe liquid enzyme concentrate and final product. Further, there is insome instances a desire for formulations which are acceptable for food,pharmaceutical, or cosmetic use, and certain salts like sodium formatemay not be acceptable or suitable for this purpose.

[0006] The use of inorganic salts as stabilization agents for enzymes isknown in the art. For instance, U.S. Pat. No. 5,460,658, Nakagawa et al,issued Oct. 24, 1995, discloses an enzyme based contact lens cleaningsolution said to be stabilized with a polyhydric alcohol (5-30%) andalkali metal salt (1-5%) combination. The Nakagawa et al. patent teachesthat polyhydric alcohol levels are less than 30% to maintain enzymeactivity in the contact lens solution application. Xylanase wasstabilized with a polyol (2040%) and formate or potassium chloride (48%)as described in a study entitled “Developmnent of A Method for theStabilization and Formulation of Xylanase from Trichoderma UsingExperimental Design”, R. Spencer Fisk and Curran Simpson, as reported inStudies in Organic Chemistry, vol. 47, Stability and Stabilization ofEnzymes, Elservier, edited by W. J. J. Van Den Tweel, A. Harder, R. M.Buitelaar, 1992. This study discloses the use of no more than 40% polyolto maintain xylanase activity.

[0007] A cosmetic formulation having an enzyme stabilized using a 30-99%water-binding polyol that is partially or totally complexed with acrylicor methacrylic polymer is described in U.S. Pat. No. 5,830,449, Afriatet al., issued Nov. 3, 1998 , and U.S. Pat. No. 5,703,041, Afriat etal., issued Dec. 30, 1997. The cosmetic formulation may also include aninorganic salt (2 to 12%) as a secondary stabilizing agent.

[0008] Clearly, there is a continuing need for liquid formulations thatcontain enzymes which are stabilized and exhibit a high activity overtime. Particularly, there is a need for protease stabilized liquidformulations that are easy to process, highly effective, inexpensive toproduce relative to the previously used stabilizing formulations,relatively inactive until ultimately included as an ingredient in aselected application, and also useful for formulations that are welltolerated physiologically.

SUMMARY OF THE INVENTION

[0009] It has been discovered that the combination of a high level ofalkali metal halide salts, such as sodium chloride (at least 4% or 5%w/w, or higher), in combination with a polyol solvent, such as glycerol,propylene glycol, sucrose, etc (30% w/w, or higher), provides a highlevel of thermal stability for protease (produced from Bacillus species,e.g., subtilisin) formulations.

[0010] One aspect of the present invention provides a method forstabilizing one or more enzymes in a liquid medium. In one embodiment,the method of the invention includes the step of formulating in theliquid medium a high level of an alkali metal halide salt in combinationwith a polyol solvent.

[0011] The alkali metal halide salt cap be, for example, sodiumchloride, potassium chloride, and sodium or potassium fluoride orbromide. Lithium salts may also be used. Additionally, other inorganicsalts, such as salts of sulfates or sulfites, carbonates, phosphates,silicates or nitrates may be used. One preferred embodiment contemplatesat least between 4-12% (e.g., at least 4%, at least 5%, at least 6%, atleast 7%, at least 8%, at least 9%, at least 10%, at least 11%, and/orat least 12%) sodium chloride. In one embodiment, the sodium chloridecomprises between about 5-12%, preferably about 8-12% w/w, of the liquidformulation.

[0012] The polyol can be present, for example, at a level of at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, and/or at least 95%. For example, one embodimentcontemplates between 50 to 98% polyol, preferably between 55 and 95%polyol, preferably between 80 to 90% polyol, and most is preferably 50to 80% polyol. Suitable polyols include, for example, glycerol,propylene glycol, sucrose, among others.

[0013] Among the various enzymes that can be stabilized by the teachingsherein, one preferred enzyme is a protease, produced from Bacillusspecies, such as a subtilisin.

[0014] The liquid medium of the invention can be, for example, a liquidenzyme concentrate or a formulated product including at least oneenzyme, such as a protease. In one embodiment, the liquid medium is aformulated product selected from the group consisting of personal careproducts, health care products, and cleaning/detergency products. Theliquid medium can be used, for example, in formulating hand creams,liquid detergents, and the like.

[0015] Another aspect of the present invention provides a liquidformulation providing an enzyme-stabilizing environment. In oneembodiment, the formulation includes one or more enzymes, a high levelof an alkali metal salt, and a polyol solvent.

[0016] The alkali metal halide salt can be, for example, sodiumchloride, potassium chloride, and sodium or potassium fluoride orbromide. Lithium salts also may be used. Additionally, other inorganicsalts, such as salts of sulfates or sulfites, carbonates, phosphates,silicates or nitrates may be used. In one embodiment, the sodiumchloride is present at a level of between 4-12% (e.g., at least 4%, atleast 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least10%, at least 11%, up to and including 12%). In an exemplaryformulation, the sodium chloride comprises about 8-12% w/w of the liquidformulation.

[0017] The polyol is preferably present, for example, at a level of atleast 30%. Certain embodiments contemplate a polyol level of at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, and/or at least 95%. One embodiment contemplatesbetween 50 to 98% polyol, preferably between 55 and 95% polyol,preferably between 80 to 90% polyol, and most preferably 50 to 80polyol. Suitable polyols include, for example, glycerol, propyleneglycol, sucrose, among others.

[0018] Among the one or more enzymes that are stabilized in theformulation, one embodiment contemplates inclusion of a proteaseproduced from Bacillus species, preferably a subtilisin.

[0019] In one embodiment, the liquid formulation is a liquid enzymeconcentrate or a formulated product including at least one enzyme, suchas a protease produced from Baccillus species, such as a subtilisin. Theliquid formulation can be, for example, a formulated product such as apersonal care product, health care product, a cleaning product, adetergency product, among others.

[0020] Still a further aspect of the present invention provides astabilized liquid enzyme formulation, including: (i) a protease, such assubtilisin; (ii) at least 5% w/w of an alkali metal halide salt, such assodium chloride, potassium chloride, sodium or potassium fluoride orbromide, or lithium salts. Additionally, other inorganic salts, such assalts of sulfates or sulfites, carbonates, phosphates, silicates ornitrates may be used; and (iii) at least 30% w/w of a polyol solvent,such as glycol. Preferably, the formulation includes a subtilisin whichis stabilized to exhibit at least about 98% activity remaining after 22days at 37 degrees C. In one embodiment, the stabilized subtilisinincludes an amino acid substitution at position 217 (e.g., Y217L).

[0021] These and other features, aspects and advantages of the presentinvention will become apparent from the following detailed description,in conjunction with the appended claims.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention provides methods for stabilizing enzymesand liquid formulations including stabilized enzymes utilizing a polyoland an alkali metal halide salt.

[0023] Percentages herein are expressed as a percentage by weight of theliquid formulation.

[0024] The inventors hereof have determined that the combination of ahigh level of an alkali metal halide salt (e.g., sodium chloride) incombination with a polyol solvent (e.g., glycol) provides a high levelof thermal stability for liquid enzyme (e.g., proteases produced fromBacillus species) formulations.

[0025] Generally, formulations of the present invention include at leastabout 30% polyol or polyhydric alcohol; for example between from about50 to about 98%, between from about 50 to about 95%, between from about80 to about 90% polyol, and/or between from about 50-80% polyol. Anexemplary liquid formulation suitable IS for inclusion in personal careapplications, as contemplated herein, includes between from about 50% toabout 80% glycerol. Other suitable polyols include, and are not limitedto, propylene glycol, ethylene glycol, sorbitol, mannitol, erythritol,dulcitol and inositol. Such a liquid formulation can be furtherformulated into, for example, a hand cream, or the like. A liquidformulation for liquid detergent applications, as contemplated herein,includes between from about 33 to about 40% propylene glycol. Anotherexemplary liquid formulation for personal care application includesabout 60% glycerol. The polyol level is approximately 50-80% to producea compound having relatively inactive enzymes until such compoundultimately is included in a specific application.

[0026] Formulations of the present invention include at least about 5%of an alkali metal halide salt, such as sodium chloride, potassiumchloride, and sodium or potassium fluoride or bromide, or lithium salts.Additionally, other inorganic salts, such as salts of sulfates orsulfites, carbonates, phosphates, silicates or nitrates may be used. Inone preferred embodiment, the alkali halide metal salt is sodiumchloride. The sodium chloride can be present, for example, at a level ofat least about 5%, at least about 6%, at least about 7%, at least about8%, at least about 9%, at least about 10%, at least about 11%, and/or atleast about 12%. In one embodiment, a liquid enzyme formulation includesbetween from about 5% to about 12% sodium chloride (e.g., preferred atabout 8% sodium chloride). In another preferred embodiment, the liquidenzyme formulation includes about 10% sodium chloride.

[0027] Preferred enzymes include those enzymes capable of hydrolyzingsubstrates. Such enzymes, which are known as hydrolases, include, butare not limited to, proteases (bacterial, fungal, acid, neutral oralkaline), amylases (alpha or beta), lipases, cellulases and mixturesthereof. Preferred proteases are also those described in US Re. 34,606and EP 0 130 756, EP 251,446, EP 451,244, and EP 775,749, andincorporated herein by reference. Other preferred proteases aredescribed in U.S. Pat. Nos. 6,218,165, 5,284,791, 5,700,676, 5,185,258,5,155,033, 5,346,823, 5,763,257 and U.S. patent application Ser. No.09/768,080, filed Feb. 8, 2000, titled Proteins Producing An AlteredImmunogenic Response And Methods Of Making And Using The Same,describing protease mutants having an altered T-cell epitope. Onepreferred protease is available under the trade name Multifect, fromGenencor International, Inc. Other preferred proteases include Purafectand Properase, from Genencor International, Inc. or Savinase, Esperase,Alcalase available from Novozymes A/S. Preferred proteases are thoseproduced from any Bacillus species and include mutants which retaintheir bacillus protease-like structure and function. Other enzymes thatcan be used in the present invention include oxidases, peroxidases,transferases, dehydratases, reductases, hemicellulases and isomerases,among others. One or more enzymes may be included in the formulations ofthe present invention.

[0028] The stabilized liquid enzyme formulations of the presentinvention can be applied in a variety of fields, including the fields ofpersonal care (e.g., protease for use in hand creams, and the like), andcleaning (e.g., protease for use in liquid detergents, and the like),among others. For example, the liquid enzyme formulations can be liquidenzyme concentrates which are useful for further formulation into finalproducts, and/or they can be final formulated products, such as skincreams, lotions, liquid soaps, liquid detergents, etc.

[0029] Liquid enzyme formulations of the present invention areparticularly effective at stabilizing enzymes during storage, within atemperature range of between from about 20 to about 40 degrees Celsius,e.g., at or around room temperature (22-25 degrees Celsius).

EXAMPLES

[0030] The following examples are illustrative and are not intended tolimit the invention.

Example 1

[0031] Subtilisin BPN′ Y217L was formulated at 5-7 gE/l in a base of80-90% glycerol, 1 mM KH2PO4 at pH 5.0. The method of formulationgenerally involves the following: the 1 mM CaCl₂ reagent was added tothe glycerol reagent followed by the addition of the selected enzyme.Water was then added and the resulting mixture was allowed to dissolveovernight at 4° C. The selected salt concentration was prepared bydissolving the salt in 1M NaOAC prior to addition to the dissolvedenzyme. Next, 3% CP carbon was added to the enzyme salt combination andthe resulting compound was mixed at 22° C. for approximately 6 hours.Next, the compound was filtered and the resulting solution was storedfor the stability studies set out below.

[0032] As shown in Table 1, the formulation was tested without furtherstabilization and with stabilization utilizing three stabilizers (sodiumacetate, sodium chloride, and sodium formate). TABLE 1 Stability Study 1Basic Conditions for all samples * 50 ppm Ca2+ 10 g enzyme solution *Temperature 25 C./37 C. Product Experiments Activity B0 KH2PO4 10 mM @pH 5.0 Sodium Acetate; 8% Glycerol; 82% 6.75 gE/l before additions; 10%B1 KH2PO4 10 mM @ pH 5.0 Sodium Chloride; 8% Glycerol; 82% 6.75 gE/lbefore additions; 10% B2 KH2PO4 10 mM @ pH 5.0 Sodium Formate; 8%Glycerol; 82% 6.75 gE/l before additions; 10% B3 KH2PO4 10 mM @ pH 5.0None Glycerol; 90% 6.75 gE/l before additions; 10% B4 KH2PO4 10 mM @ pH5.0 None Glycerol; 90% 3.37 gE/l before additions; 10% B5 KH2PO4 10 mM @pH 5.0 None Glycerol; 45%/Propylene 6.75 gE/l before additions; 10%Glycol; 45% B6 H2PO4 10 mM @ pH 5.0 None Propylene Glycol; 90% 6.75 gE/lbefore additions; 10% B7 KH2PO4 10 mM @ pH 5.0 None Glycerol; 90% 6.75gE/l after additions; 10% B8 KH2PO4 10 mM @ pH 5.5 None Glycerol; 90%6.75 gE/l before additions; 10%

[0033] Activity levels for all of the above formulations were monitoredas exhibited by the data of Table 2, which demonstrates that sodiumchloride is a very effective agent for stabilization of subtilisin(note, in particular, B 1). Remaining enzyme activity at 22 daysutilizing the three stabilizers as shown was between 96-98% as comparedto a value of only 81% in the remaining formulations that did notinclude sodium acetate, sodium chloride or sodium formate. TABLE 2 37°C. Subtilisin Stability Study Activities by AAPF-pNA Assay in ΔAUmin⁻¹ml⁻¹ Buffer Day 0 1 2 3 4 5 6 7 8 0 206 211 233 229 112 234 204 200204 1 212 216 239 238 113 238 214 171 214 6 205 210 225 214 98 217 177161 198 22 196 206 224 185 49 179 109 142 160 41 158 208 212 150 11 16362 116 138 66 186 205 202 129 7 132 32 33 113 94 183 192 183 88 Activityleft %  Buffer Day B0 B1 B2 B3 B4 B5 B6 B7 B8 0 100 100 100 100 100 100100 100 100 1 103 102 102 104 101 102 105 85 105 6 100 100 98 94 88 9387 80 97 22 96 98 96 81 43 77 54 71 78 41 91 99 91 66 10 70 30 58 67 6691 97 87 57 6 57 16 16 55 94 89 91 79 43

[0034] The 8% NaCl sample was found to have high stability (over 98%activity remaining after 22 days at 37 degrees C. and 100% after 23 daysat 37 degrees C.). The data further demonstrates that a high activitylevel (at least 90%) remained in the presence of sodium chloride out to94 days.

[0035] Table 3 below represents the same example as above conducted at25° C. TABLE 3 25° C. Subtilisin Stability Study Activities by AAPF-pNAAssay in ΔAU min⁻¹ml⁻¹ Buffer Day 0 1 2 3 4 5 6 7 8 0 206 211 233 229112 234 204 200 204 2 205 216 223 227 108 227 200 184 197 23 204 212 242214 94 223 188 166 203 30 200 213 220 214 85 215 182 153 197 65 201 206227 192 40 187 158 135 174 94 189 197 222 158 Activity left %  BufferDay B0 B1 B2 B3 B4 B5 B6 B7 B8 0 100 100 100 100 100 100 100 100 100 2100 102 96 99 97 97 98 92 96 23 99 101 104 93 84 95 93 83 99 30 97 10194 93 76 92 90 77 97 65 98 98 97 84 35 80 78 68 77 94 92 93 95 77

[0036] Example 2 illustrates the results of a study designed to increasethe commercial desirability of liquid enzyme formulations and tofacilitate manufacturing of such formulations. Accordingly, Example gillustrates the surprising results achieved by reducing theconcentration of glycerol and increasing the concentration of salt inthe formulation.

[0037] Subtilisin BPN′ Y217L was formulated as set out in Table 4 belowin a base of 70-82% glycerol, 1 mM KH2PO4. TABLE 4 Stability Study 2Basic Conditions for all samples * 50 ppm Ca2+ 10 g enzyme solution *Temperature 25 C./37 C. Target Enzyme Final Enzyme Salt** Glycerol**Activity Enzyme Form Form # Buffer (g) Salt Type (g) (mg/mL) (wt/wt %)Density B1  10 mM 8 NaOAC 82 6.75 0.54 1.25 K-Phos, ph 5.0 B2  10 mM 8NaCl 82 6.75 0.54 1.25 K-Phos ph 5.0 B3  10 mM 8 NaOAC.3H2O 82 1.3 0.1041.25 K-Phos ph 5.0 B4  10 mM 12 NaOAC.3H2O 70 1.3 0.104 1.25 K-Phos ph5.5 B5  10 mM 8 NaCl 82 1.3 0.104 1.25 K-Phos ph 5.5 B6  10 mM 6 NaCl 821.3 0.104 1.25 K-Phos ph 5.5 B7  10 mM 8 NaCl 70 1.3 0.104 1.25 K-Phosph 5.5 B8  10 mM 10 NaCl 60 1.3 0.104 1.25 K-Phos ph 5.5 B9 100 mM 6NaCl 62 1.3 0.104 1.25 K-Phos ph 5.5 B10 100 mM 8 NaCl 62 1.3 0.104 1.25K-Phos ph 5.5 B11 100 mM 8 NaCl 70 1.3 0.104 1.25 K-Phos ph 5.5 B12 100mM 10 NaCl 60 1.3 0.104 1.25 K-Phos ph 5.5 B13 100 mM 8 NaOAC.3H2O 821.3 0.104 1.25 K-Phos ph 5.5 B14 100 mM 12 NaOAC.3H2O 70 1.3 0.104 1.25K-Phos ph 5.5

[0038] As shown by the surprising data of Table 5 below, a stableglycerol/salt formulation that is easily manufactured may be achieved byincreasing the salt concentration thereby enabling a reduction in theamount of glycerol in the formulation. Table 5 illustrates that a 60%glycerol and 10% salt formulation is exceptionally stable. TABLE 5Subtilisin Stability Study: 25° C. Activities by AAPF-pNA Assay in ΔAUmin⁻¹ml⁻¹ Buffer Day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 323 327 63.363.7 63.8 66.4 63.4 66.5 65.3 66.9 65.4 63.5 64.9 63.2 118 310 327 63.661.4 66.9 60.9 61.7 66.9 60.9 63.2 66.4 64.4 63.8 62.6 174 320 336 65.963.7 62.6 59.1 63.6 68.6 61.7 64.6 64.1 66.8 65.6 63.7 279 301 301 57.759.9 51.8 50.8 62.6 62.1 53.8 54.8 59.9 59.4 57.4 59.0 % OriginalActivity B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 0 100 100 100100 100 100 100 100 100 100 100 100 100 100 118 96.0 100.0 100.5 96.4104.8 91.6 97.3 100.7 93.3 94.5 101.6 101.4 98.2 99.0 174 99.2 102.6104.2 100.1 98.1 88.9 100.2 103.1 94.6 96.5 98.0 105.1 101.1 100.9 27993.2 91.8 91.2 94.0 81.2 76.5 98.6 93.3 82.5 81.9 91.6 93.5 88.4 93.4

[0039] TABLE 6 37 C Activities by AAPF-pNA Assay in ΔAU min⁻¹ml⁻¹ BufferDay 1 2 3 4 5 6 7 8 9 10 11 12 13 14 0 323 327 63.3 63.7 63.8 66.4 63.466.5 65.3 66.9 65.4 63.5 64.9 63.2 9 313 316 63.1 64.0 64.3 64.2 62.763.0 61.6 64.1 60.7 61.4 62.4 61.2 29 311 320 63.3 63.2 64.1 63.4 62.364.4 60.4 64.3 61.1 61.7 62.7 61.4 112 269 298 57.9 55.3 62.4 48.4 64.763.4 48.9 62.2 60.9 58.9 57.4 56.5 173 281 233 54.7 50.3 57.3 12.4 61.764.6 27.7 60.7 61.7 55.9 52.8 52.6 280 242 43.9 48.3 44.6 6.79 4.42 56.958.3 4.76 32.3 52.5 55.5 47.1 42.4 % Original Activity Buffer Day B1 B2B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 0 100 100 100 100 100 100 100100 100 100 100 100 100 100 9 97.0 96.6 99.8 100.5 100.7 96.9 98.9 94.794.4 95.8 92.8 96.7 96.2 96.8 29 96.4 97.7 100.0 99.2 100.4 95.4 98.196.9 92.5 96.0 93.4 97.2 96.7 97.1 112 83.4 90.9 91.4 86.9 97.8 72.9102.0 95.4 75.0 93.0 93.2 92.7 88.4 89.4 173 87.0 71.2 86.4 79.0 89.818.7 97.3 97.1 42.5 90.7 92.9 97.2 86.1 83.3 280 75.1 13.4 76.3 70.010.6 6.6 89.6 87.7 7.3 48.3 80.3 87.4 72.6 67.1

Protease Assay

[0040] Examples 1 and 2 measured activity utilizing a protease assaywith N-succinyl-L-Ala-L-L-Ala-L-Pro-L-Phe-p-nitroanilide (suc-AAPF-pNA).The assay is based upon the cleavage by proteases of the amide bondbetween phenylalanine and p-nitroaniline of the N-succinyl reagent.P-nitroaniline is monitored spectrophotometrically at 410 nm and therate of the appearance of p-nitroaniline is a measure of proteolyticactivity. A protease unit is defined as the amount of protease enzymethat increases absorbance at 410 nm by 1 absorbance unit (AU)/min of astandard solution of 1.6 mM suc-AAPF-pNA in 0.1 M Tris Buffer at 25° C.in a cuvet with a 1 cm path length. The glycerol was obtained from JTBaker.

[0041] Various other examples and modifications of the foregoingdescription and examples will be apparent to a person skilled in the artafter reading the disclosure without departing from the spirit and scopeof the invention, and it is intended that all such examples ormodifications be included within the scope of the appended claims. Allpublications and patents referenced herein are hereby incorporated byreference in their entirety.

1. A method for stabilizing one or more protease enzymes produced fromBacillus species in a liquid medium, comprising: formulating in saidliquid medium a high level of an alkali metal halide salt in combinationwith a polyol solvent.
 2. The method of claim 1, wherein said alkalimetal halide salt comprises sodium chloride, said sodium chloride beingpresent at a level of at least about 4%, preferably at least about 5%,and most preferably between from about 5% to about 12%.
 3. The method ofclaim 1, wherein said polyol is present at a level of at least about50%, preferably between from about 50 to about 80%.
 4. The method ofclaim 1, wherein said polyol solvent is selected from the groupconsisting of glycerol, propylene glycol, sucrose, and any combinationthereof.
 5. The method of claim 1, wherein said one or more proteaseenzymes is a subtilisin.
 6. The method of claim 1, wherein said liquidmedium is a liquid enzyme concentrate or a formulated product.
 7. Themethod of claim 6, wherein said liquid medium is a formulated productselected from the group consisting of personal care products, healthcare products, cleaning products, and detergency products.
 8. A liquidformulation providing an enzyme-stabilizing environment, saidformulation including one or more proteases produced from a Bacillusspecies, a high level of an alkali metal halide salt, and a polyolsolvent.
 9. The formulation of claim 8, wherein said alkali metal saltcomprises sodium chloride, said sodium chloride being present at a levelof at least about 4%, preferably at least about 5%, and most preferablybetween from about 5% to about 12%.
 10. The method of claim 8, whereinsaid polyol is present at a level of at least about 50%, preferablybetween from about 50 to about 80%.
 11. The formulation of claim 10,wherein said polyol solvent is selected from the group consisting ofglycerol, propylene glycol, sucrose, and any combination thereof. 12.The formulation of claim 8, wherein said one or more protease enzymesincludes a subtilisin.
 13. The formulation of claim 8, wherein saidliquid formulation is a liquid enzyme concentrate or a formulatedproduct.
 14. The formulation of claim 13, wherein said liquidformulation is a formulated product selected from the group consistingof personal care products, health care products, cleaning products, anddetergency products.
 15. A stabilized liquid enzyme formulation,including: (i) a protease produced from any Bacillus species, (ii) atleast about 5% of an alkali metal halide salt, and (iii) at least about50% of a polyol solvent.
 16. The formulation of claim 15, wherein saidalkali metal salt comprises sodium chloride.
 17. The formulation ofclaim 15, wherein said polyol solvent comprises propylene glycol. 18.The formulation of claim 15, wherein said protease has at least about95% activity remaining, preferably at least about 98% activityremaining, after 22 days at 37 degrees C.
 19. The formulation of claim15, wherein said protease is a subtilisin.
 20. The formulation of claim19, wherein said subtilisin includes an amino acid substitution atposition 217.